Project summary Physical pressure is fundamentally important for cancer biology, but its effects remain poorly understood. When solid tumors grow confined within surrounding tissue, they build up compressive stress. Given that cells evolved to function in a stable mechanical environment, even slight changes in pressure perturb physiology. Normal cells and early stage cancer cells stop growing when pressure builds up. In contrast, in advanced cancer, compression can change cellular behavior to drive migration of cancer cells to other organs or confer resistance to chemo- therapy. This difference implies that cancer cells somehow adapt to physical pressure. A lack of tools has slowed progress in understanding the relationships between compression, the physical properties of cells, and cancer behavior. We developed two new technologies to overcome this limitation: First, we created a gene that enables cells to produce a steady supply of fluorescent nanoparticles that act as tell-tales for shifts in intracellular physical properties. Second, we developed microfluidic devices to control compressive stress, either quickly or slowly, while maintaining a constant chemical environment. We will combine these innovations to test the overarching hypothesis that mutations that confer resistance to mechanical compression enable pancreatic cancer cells to adapt to their high-pressure environment and drive their oncogenic evolution. Aim 1: We will determine how compression differentially impacts wildtype and mutant pancreatic cells. We will use GEM nanoparticles to quantify the physical response to pressure and test the hypothesis that oncogenic mutations alter both the physical and physiological response to pressure. Aim 2: We will determine the effects of compression on phase separation. We will investigate the hypothesis that decreased cell volume under pressure leads to in- creased phase separation of stress granules. We will evaluate molecular crowding as a mechanism for these effects. We will determine the importance of stress granule formation for mechanical adaptation and drug re- sistance. Aim 3: We will determine genetic mechanisms of pressure adaptation. We will follow up on pre- liminary mutants that confer resistance to compression, using a CRISPR modifier screen to determine mecha- nisms of adaptation. We will overexpress known oncogenes to find further adaptation pathways. Our innovative combination of genetic nanoparticles and microfluidic approaches, and our expertise that bridges biophysics, mechanobiology and cell biology make us uniquely qualified to connect compression, the physicochemical properties of cells, and cancer physiology. Our studies promise to reveal key network pertur- bations essential to cancer cell growth and survival under pressure. Understanding these adaptive mechanisms promises to suggest treatments that exploit the aberrant mechanical properties of tumors caused by high com- pressive stress.